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DIAMETRIXDOWNREGULATESINFLAMMATORYSIGNALINGINOBESETYPE2DIABETES MURINEMODELINVIVO KennethR.Hampshire1,StevenMiles1,RickMiles1,RobertT.Streeper2,ArmandoDiaz2,David Campos2,JoelMichalek3,ChristopherLouden3,WieslawFurmaga3,ElzbietaIzbicka2&

1 2

SyntratechCorporation,Denver,Colorado,USA CPCLTD,SanAntonio,Texas,USA 3 UniversityofTexasHealthScienceCenteratSanAntonio,SanAntonio,Texas,USA & correspondingauthor ABSTRACT Background:DiaMetrix,adietarysupplement,loweredbloodglucoseandimprovedseveral surrogateendpointsinaclinicalstudyofdiabeticpatients.Thisstudycomparedtheactivityof DiaMetrixtothatofantidiabeticdrugsinanobesediabeticmousemodel. Methods:DiaMetrix,metformin,Actos(pioglitazonehydrochloride),andByetta(exenatide) wereadministeredtoBKS.Cgm+/+Leprdb/BomTacfemalemice.Untreatedanimalsservedasa control.Animals(8pergroup)werefedeithernormaldiet(ND)orhighfatdiet(HFD)+/drugs for8weeks.Bodymasswasmeasuredweekly.Wequantifiedplasmalevelsof40biomarkers (chemokines,cytokines,endocrine,growthfactorsandmetabolites)includinginsulin,glucose, advancedglycationendproduct(AGE),cholesterolandtriglycerides.Pyruvatekinaseactivity, citrate,ADPandATPconcentrationsandhexokinaseIIexpressionweredeterminedinmuscle tissue.Organpathologywasassessedmicroscopically.Meanvaluesofall45biomarkerswere comparedbetweentreatmentgroups. Results:DiaMetrixsprotectionagainstorgandamagewascomparabletothatofByettaand betterthanActosandmetforminintheanimalsonND.Themeanvaluesofmostplasma biomarkerswereelevatedinHFDrelativetoND.Biomarkermeansvariedsignificantlyby treatmentgroupanddiet.OnND,DiaMetrixdecreasedlevelsofeotaxin,MCP1,MCP3,M CSF,andincreasedIL4relativetountreated.DiaMetrixtreatmentdecreasedGCSF,GMCSF, andTGFrelativetountreatedinHFDanimals.PyruvatekinaseandAGEincreased,while insulinwasdecreasedinanimalstreatedwithDiaMetrixrelativetountreatedonND. Treatmentgroupcontrastsonbiomarkers(MCP3,IL17,AGE,andinsulin)alsovariedwithdiet. Conclusions:DiaMetrixdemonstratedsuperiorantiinflammatoryactivityrelativetothe commonlyusedantidiabeticdrugsagainstabackgroundofgeneticobesity.Theseresults, takentogetherwiththeresultsofhistologicalexamination,supportthecontentionthat DiaMetrixmaybeaneffectiveinterventionfortype2diabetes.

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Introduction Diabetesmellitustype2,alsoknownastype2diabetes,isanautoimmune inflammatorydisease,whichisfueledbypathologicalexpressionoftheinnateimmunesystem innonimmunehypothalamiccells,visceraladipocytes,cellsofthepancreas,andvascular endothelium(1).Amongriskfactors,ahighfatdietisconsideredtobedetrimentaltopeople withdiabetes.Consumptionofafatrichdietactivatesaproinflammatoryresponseand inducesinsulinresistanceinthehypothalamus(2),(3).Dietaryfatcauseshighbloodsugar levelsbypreventingsugarfromenteringmusclesandothercells,thuskeepingsugarinthe bloodstreamandelevatingittohighlevels(4),(5).Inaddition,physicalinactivityandobesity aretwomajorriskfactorsforthedevelopmentoftype2diabetes(6).Highbloodglucosein thecontextofinsulinresistanceandrelativeinsulindeficiencyisassociatedwithdecreased ratesofglycolysis,glycogenesis,lipogenesis,andproteinsynthesis.Undernormalconditions, morethan80%oftheenergyproducedbythebodyisderivedfromcarbohydratemetabolism. Whenthismetabolismisseverelylimited,thecellinitiatesoxidationoffatreservesforenergy productionanddegradesproteinstoaminoacidswhichinturnareconvertedtoglucose.If excessivefatmetabolismoccursinconjunctionwithinadequatecarbohydrateutilization,there areinadequateamountsofoxaloaceticacidwithwhichtoreactwithacetylCoAfromthefatty acidoxidation.AnexcessofacetylCoAleadstoabuildupofketonebodiesleadingtoketosis andacidosis.Longtermcomplicationsofthediabeticconditionincludearteriosclerosisand othercardiovascularproblems,changesintheretinaandtheformationofcataracts,nervous systemproblemsandkidneydiseases. DiaMetrixisanherbaldietarysupplementcomposedofvitaminC(ascorbicacid), biotinUSP,chromium(chelate),vanadium(chelate),garciniacambogiaextract(50% hydroxycitricacid),gymnemasylvestreextract(25%),cinnamonextract(4:1),bittermelon extract(10:1),betaineHCL,banabaextract(1%corosolicacid),fennugreek,dicalcium phosphate,cellulose,croscarmellosesodium,stearicacid,silicondioxide,magnesiumstearate, andhydroxypropylmethylcellulose.Arecentdoubleblindedclinicalstudyofdiabeticpatients demonstratedthatDiaMetrixloweredbloodglucoselevels,triglycerides,cholesterol,andblood pressure(Hampshireetal,manuscriptinpreparation).ToshedmorelightonDiaMetrixsmode ofaction,wecomparedtheactivityofDiaMetrixtothreecommonlyusedantidiabeticdrugs: metformin,anantihyperglycemicagentthathasbeeninusefornearlytwodecades(7)and twomorerecentdrugs;Actos(pioglitazonehydrochloride)(8)andByetta(exenatide)(9). Thesedrugswereexaminedatdosescomparabletothoseusedinclinicalpracticeinamurine modelusingBKS.Cgm+/+Leprdb/BomTac(db/db)femalemice.Thesemicearehomozygousfor apointmutationinthegenefortheleptinreceptorLEPR(10),aproteinthatinhumansis encodedbytheLEPRgene.Thedb/dbmouseisamodelofobesity,diabetes,anddyslipidemia (11)(12).LEPRdeficientdb/dbmicedevelopdiseasesimilartohumantype2diabetes mellitus,hypertension,andobesitywithdisruptedcircadianbloodpressurerhythm.Theyare alsoknowntodevelophyperglycemiaatapproximately6weeksageandfrankimmune dysfunctionby20weeks(13).

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Usingthisinvivosystem,weselectedabroadrangeofendpointstoevaluate mechanismsofactionofDiaMetrix.Inadditiontocommonlytestedmetabolites(glucose, advancedglycationendproduct,insulin,cholesterol,triglycerides),weincludedpyruvate kinase,hexokinaseII,andcitrateaspotentiallyrelevantendpointsalongwithalargepanelof cytokines,chemokines,endocrinemarkersandgrowthfactors. Methods Animals:Thestudywasperformedaccordingtotheguidelinesforthecareanduseof laboratoryanimalsatCPCLLC,SanAntonio,Texas.BKS.Cgm+/+Leprdb/BomTacfemalemice thatwere7to9weeksoldatshipmentweresourcedfromTaconicFarms(Hudson,NY).The animalswerehouseduntil10to12weeksofagebeforestartoftreatment. Animaldietanddrugtreatment:Animalswererandomizedintotwogroupsof40miceeach andfedadlibitumwithPurina5001rodentdiet(normaldiet;ND)orBioServHighFatDietp.n. F1850(BioServ,Frenchtown,NJ08825)rodentdietpaste(highfatdiet;HFD).Eachdietgroup wasfurtherdividedintofivetreatmentgroups(Actos,Byetta,metformin,DiaMetrix,untreated) with8animalsforeachtreatmentgroup. TherecommendeddosageofDiaMetrixissixcapsulesperday.Thus,ifwerefertothe maximumaverage75thpercentilebodymassofwhitefemalesat82kgthedailydosageis6X 1600mg/82kg=117.12mg/kg.TheaveragebodymassofBKS.Cgm+/+Leprdb/BomTacmiceis ~20g(0.02kg)at9to12weeksofage.Theaveragedailyfoodconsumptionofananimalof thissizeis~15gm/day.Thus,117.12mgx0.02kg=2.3424mg/kg/dayofDiaMetrixin15gm offoodforafinalconcentrationof156.12mgDiaMetrix/kgofrodentdiet.Thisdietwas preparedbyblendingtheappropriatequantitiesofDiaMetrixwithHFD.Anequivalentdrug formulationinNDwaspreparedbyusingwatertosoftenthefoodgranulesreducingthemto pasteintowhichthevariousoraldrugswereincorporatedbymechanicalmixing.Metformin wasformulatedat0.12%byweightinthetwofeedpreparations.Actostypicaldailydosageis4 mg/dayandthedrugwasformulatedat0.065mg/kginthetwodiets.Byettawasdilutedin PBStoafinalconcentrationof12.2ng/mL.Theresultingsolutionwasadministeredby subcutaneousinjection(100L )twiceperday,onceinthemorningandonceintheafternoon. Tissueprocessing:Attheterminationoftheexperiment,atapproximately60days,theanimals wereeuthanizedandexsanguinated.WholeperipheralbloodwascollectedintoBDVacutainer CPTCellPreparationTubes(BecktonDickinson,FranklinLakes,NJ)andcentrifugedat1,500xg atroomtemperaturetoisolateplasmaaccordingtosuppliersprotocol.Dissectedorgans (eyes,kidneys,muscle,pancreas,smallandlargeintestine)werefixedin10%phosphate bufferedformalin(FisherScientific,Pittsburgh,PA).Tissuespecimenswereembeddedin paraffinandstainedwithhematoxyllinandeosin.TissueimagesweregeneratedintheCore OpticalImagingFacility,whichissupportedbyUniversityofTexasHealthSciencesCenteratSan Antonio,NIHNCIP30CA54174(CTRC),NIHNIAP30AG013319(NathanShockCenter)andNIH NIAP01AG19316.

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Bioassays:Theplasmawasanalyzedbymultiplexedimmunoassayforthequantitative determinationof34cytokines,chemokinesandgrowthfactorsusingProcartacytokineprofiling kits(Affymetrix,Fremont,CA).Advancedglycationendproduct(AGE)OxiSelectELISA(#STA 317)waspurchasedfromCellBioLabs,SanDiego,CA.InsulinELISAkit(rat/mouse,#EZRMI 13K)wasobtainedfromLincoResearch,St.Charles,MO.Assaykitsforglucose(#K613), pyruvatekinase(#K709),citrate(#K655),HDLandLDL/VLDLcholesterol(#K709),triglycerides (#K622),andAposensorADP/ATPratiowereobtainedfromBioVision(MountainView,CA). Forcitrateassay,plasmawasdeproteinizedusingasamplepreparationkitfromBioVision (#K808). Statisticalanalysis:Continuouslydistributedoutcomesweresummarizedwiththemeanand standarddeviation.Relativevaluesweregraphicallyrepresentedwithaheatmap.Ateach levelofdiet(normal,highfat),treatmentgroupswerecontrastedwithregardtothemean usinganalysisofvarianceandforeachbiomarker,pairwisecomparisonsbetweentreatment groupswerecorrectedformultipletestingusingtheTukeymethod.TheHochbergcorrection wasappliedacrossallbiomarkerswithineachlevelofdiettocorrectformultipletesting. Treatmentgroupcontrastswithregardtobodymasswerebasedonarepeatedmeasures linearmodelwithanautoregressiveorder1covarianceassumptionintermsoftreatment,day, andthetreatmentbydayinteraction.Allstatisticaltestingwastwosidedwithanominaland experimentwisesignificancelevelof5%usingSASVersion9.2.Rwasusedforgraphics. Results Bodymass:Animalbodymass(g)measuredthroughthecourseofthestudyaresummarized bytreatmentgroupinFigures1(normaldiet)and2(highfatdiet).Inanimalsfedanormaldiet bodymassesincreasedcontinuouslybothincontrolanimalsandalldrugtreatmentgroups.In contrast,bodymassesofallmicemaintainedonhighfatdietinitiallyincreasedthroughfirst34 weeksthenbegantograduallydecline.Thesacrificetimewasdeterminedbasedonanimal appearanceandperformance,especiallyinthehighfatdietmice.Miceinthecontroland ByettatreatmentgroupsweresacrificedonDay63.AnimalstreatedwithDiaMetrixwere terminatedonDay56,whilemetforminandActostreatedmiceweresacrificedonDay49 becauseoftheirlargelycompromisedperformance.TherewasoneprematuredeathinByetta animalsonnormaldietandtwodeathsinDiaMetrixtreatedanimalsonhighfatdiet.There werenosignificantdifferencesbetweenthecurvesineitherdiet. TreatmentgroupcontrastswithDiaMetrixandDiaMetrixcontrastswithuntreatedon meanweightattheendofstudywerenotsignificant,regardlessofdiet[Normaldiet(Actos: 48.74.8,Byetta:52.85.9,metformin:49.33.9,DiaMetrix:47.38.0,untreated:51.12.7) Highfatdiet(Actos:52.02.3,Byetta:44.62.0,metformin:49.84.0,DiaMetrix:44.94.1, untreated:42.15.9). Necropsy:Controlmiceonnormaldietappearedhealthywithnovisiblewoundsorscarring, goodskinturgorandnormalactivitylevel.Largeamountsoffatwereseeninviscera.Liver, lungs,kidneys,pancreas,cardiactissueandintestineswereunremarkable.Rearlegmuscles

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hadsomeatrophy.Controlmiceonhighfatdietappearedunhealthy,lethargicandnonreactive toenvironmentalstimuli.Ecchymosiswasnotedattheinjectionsites.Viscerawerecovered withabundantfattissue.Pancreaswasyellowincolorandsmallerthanonnormaldiet.The intestineswereyellowishandswollen. Metformintreatedmiceonnormaldietwerehealthywithnovisiblewoundsorscarring, hadgoodskinturgorandnormalactivitylevel.Therewerenoremarkabledifferencesin comparisonwithrespectivecontrol,exceptformottledliverobservedinmostanimals. Metformintreatedmiceonhighfatdiethadlargeskinlesionsandextensivehairloss.Skinhad goodturgor.Theanimalswerelethargicandnonreactivetoenvironmentalstimuli.Viscera wereyellowishandtheliverhaddepositsofyellowadiposetissue.Thepancreaseswere enlarged.Therewerediffusetissuehemorrhages.Theanimalshadpoorbloodclotting. Severalanimalshadkidney/adrenaltumors. AllActostreatedmiceonnormaldietappearedhealthybuthaddecreasedactivitylevel. Thekidneyslookedhealthybutwereencapsulatedinfatandshowedhemorrhaged vasculature.Liversweremottledinmostanimals.Largeandsmallintestineswerereddishin color.MicetreatedwithActosplushighfatdietwereclearlylethargic.Alargeamountof densefatwasseenwithintheupperperitonealandretroperitonealspace.Kidneyswere enlargedandencapsulatedinadiposetissue.Thepancreaswaspaleandenlarged.Theliver hadsmalldepositsofyellowadiposetissue.Thelargeandsmallintestineswereyellowand swollen.Theanimalshadsomewastingofskeletalmuscleandgrosssteatohepatitsevidenced bydiffuseyellowspotting.Theanimalsdidnotbleedeasily. MicetreatedwithByettaandmaintainedonnormaldietwerelessactivethanthe respectivecontrols.Theirvisceralookedhealthybutthekidneyswereenlargedandthe spleenswerenecroticinmanycases.Thepancreaswasenlargedandsclerosedbuthadnormal colorandshape.Theliverwasmottledwithfattydeposits.Othertissueswereunremarkable. Byettatreatedmiceonhighfatdietlookedunhealthy,lethargic,hadecchymosisatthe injectionsites,poorskinturgorandmultipleheadandnecklesions.Themajorityofanimals showedsignsofabdominalaorticaneurysm.Thekidneyswereenlargedandencapsulatedin fatwithleakybloodvessels.Thepancreaswaspoorlyperfused,paleincolor,reducedinsize andsclerosed.Boththeliverandintestineswereyellow.Thelivershadsmalldepositsoffat andtheintestineswereswollen.Theanimalshadhairlossandedemawithsomescabbingat injectionsitesindicativeofpoorwoundhealing. TheDiaMetrixtreatedmiceonnormaldiethadnovisiblewoundsorscarringand displayedgoodskinturgor.Theiractivitylevelwassomewhatdiminishedincomparisonwith controls.Thespleen,intestines,lungsandheartwereunremarkable.Thekidneysand pancreaswereenlarged.Theliversweremottledandnecroticwithfattydepositsinmost animals.MiceonhighfatdietthatweretreatedwithDiaMetrixlookedunhealthyand lethargicwithvisibleecchymosedtissuealongthespineprimarilyattheinjectionsites,poor skinturgorandmultiplelesionsintheheadandneckregion.Visceraltissuewaspoorly oxygenated.MostorganappearancewassimilartothatofDiaMetrixplusnormaldiet.Some

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miceshoweddistinctsignsofabdominalaorticaneurysm.Smallandlargeintestineswere yellowandswollen.DiaMetrixtreatedanimalshadhairlossandedemabutnotasextensiveas theanimalstreatedwithByetta. Overall,basedonappearanceoftheanimals,drugefficacyfollowedtheorder: DiaMetrix>Byetta>Actos>metformin. Biomarkerdifferencesbytreatmentgroupanddiet Biomarkermeansvariedsignificantlybytreatmentgroupanddiet(Tables1and2)andthe meanvaluesofmostbiomarkerswereelevatedinanimalsonhighfatdietrelativetonormal diet. Cytokines,chemokines,endocrinemarkersandgrowthfactors Withthenormaldietthemeaneotaxin(ng/mL),MCP1(pg/mL)andMCP3(pg/mL)and MCSF(pg/mL)weredecreasedaftertreatmentwithDiaMetrixrelativetountreated[Eotaxin (DiaMetrix:2.70.5,Untreated:5.31.9;p=0.005),MCP1(DiaMetrix:51.216.7,Untreated: 94.618.4;p<0.001),MCP3(DiaMetrix:224.198.3,Untreated:52672.6;p<0.001),MCSF (DiaMetrix:53.1,Untreated:9.32.4;p=0.04)]andwiththehighfatdietanimalsthesemean valuesweredecreasedafterwithDiaMetrixbutnotsignificantlyso.

ThemeanofIL4(pg/mL)wasincreasedaftertreatmentwithDiaMetrixrelativeto untreatedwithbothdietsbutthisincreasewassignificantonlywiththenormaldiet[Normal diet(DiaMetrix:2.62.0,Untreated:0.80.5;p=0.04),HighFatDiet(DiaMetrix:2.02.2, Untreated:0.70.6;p=0.22)]. ThemeanofthegrowthfactorsGCSF,GMCSF,andTGFweresignificantlydecreased inanimalstreatedwithDiaMetrixrelativetountreatedinthehighfatdiet[NormaldietGCSF (DiaMetrix:73.287.2,Untreated:44.340.1;p=1.0),GMCSF(DiaMetrix:182.4256.6, Untreated:226.6535.2;p=0.98),TGF(DiaMetrix:54.825.6,Untreated:83.674.9;p=0.93), HighFatDietGCSF(DiaMetrix:56.274,Untreated:735.8769.6;p=0.02),GMCSF(DiaMetrix: 81.9129.7,Untreated:457.8433.1;p=0.03),TGF(DiaMetrix:22.534.3,Untreated: 74.972.1;p=0.005)]. Metabolites Themeanpyruvatekinase(mU/mL)wasincreasedinanimalstreatedwithDiaMetrix relativetountreatedinbothdiets,butsignificantlysowiththenormaldiet[Normaldiet (DiaMetrix:2.10.9,Untreated:0.70.9;p=0.02)HighFatDiet(DiaMetrix:2.00.8,Untreated: 1.60.8;p=0.94)].

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Themeanadvancedglycationendproduct(mg/dL)(Figures3and4)wasincreasedafter treatmentwithDiaMetrixrelativetountreatedwithbothdietsandtheincreasewassignificant onlywiththenormaldiet[Normaldiet(DiaMetrix:5.42.1,Untreated:1.60.6;p<0.001)High FatDiet(DiaMetrix:2.41.8,Untreated:1.70.8;p=0.90)]. Themeaninsulin(ng/mL)(Figures5and6)wassignificantlydecreasedaftertreatment withDiaMetrixrelativetountreatedwiththenormaldietandnonsignificantlyincreasedwith thehighfatdiet[Normaldiet(DiaMetrix:4.44.9,Untreated:16.86.0;p<0.001)HighFatDiet (DiaMetrix:3.51.3,Untreated:3.04.6;p=0.55)]. Themeancitrate(mM)(Figures7and8)wasincreasedaftertreatmentwithDiaMetrix withbothdiets,significantlysowiththehighfatdiet[Normaldiet(DiaMetrix:0.40.24, Untreated:0.210.19;p=0.09)HighFatDiet(DiaMetrix:0.630.16,Untreated:0.220.27; p=0.007)]. Treatmentgroupcontrasts Treatmentgroupcontrastsonbiomarkermeansvariedwithdietandbiomarker.With thenormaldiet,themeanMCP3(pg/mL)wassignificantlyincreasedaftertreatmentwith Actos,ByettaandmetforminrelativetoDiaMetrix[Actos:460.7189.9(p<0.001),Byetta: 476.099.9(p<0.001),metformin:502.7175.8(p<0.001),DiaMetrix:224.198.3];the correspondingcontrastswerenotsignificantwiththehighfatdiet[Actos:757.8193.2 (p=0.81),Byetta:860.5177.5(p=0.57),metformin:457.0287.7(p=0.94),DiaMetrix: 455.7433.1]. Withthehighfatdiet,themeanIL17(pg/mL)wasdecreasedaftertreatmentwith DiaMetrixrelativetoActos,Byettaandmetformin,significantlysorelativetoByetta[Normal diet,Actos:66.241.5(p=0.51),Byetta:133.053.5(p=0.81),metformin:144.5127.7(p=0.91), DiaMetrix:104.654.8,Highfatdiet,Actos:439.0230.0(p=0.09),Byetta:953.4660.5 (p<0.001),metformin:377.2181.2(p=0.18),DiaMetrix:196.4159.8]. Withthenormaldiet,themeanadvancedglycationendproduct(mg/dL)was significantlydecreasedaftertreatmentwithActosandmetforminrelativetoDiaMetrix[Actos: 1.60.3(p<0.001),Byetta:4.41.8(p=0.79),metformin:1.90.7(p<0.001),DiaMetrix:5.42.1]; thecorrespondingcontrastswerenotsignificantwiththehighfatdiet[Actos:2.51.9(p=1.0), Byetta:2.82.5(p=1.0),metformin:1.30.2(p=0.64),DiaMetrix:2.41.8]. Withthenormaldiet,themeaninsulin(ng/mL)wassignificantlyincreasedafter treatmentwithActos,ByettaandmetforminrelativetoDiaMetrix[Actos:8.84.5(p=0.04), Byetta:11.64.9(p=0.004),metformin:20.72.5(p<0.001),DiaMetrix:4.44.9];the correspondingcontrastswerenotsignificantwiththehighfatdiet[Actos:4.43.6(p=1.0), Byetta:1.40.4(p=0.73),metformin:4.64.1(p=1.0),DiaMetrix:3.51.3].

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Bivariateclusteringbytreatmentgroupandbiomarkerwascarriedoutforeachdietand summarizedwithheatmaps(Figures9and10);inthesefigureshighlevelsofabiomarkerare representedinblue,lowlevelsingreenandmissingvaluesareindicatedwithwhite. ComparisonofFigure9(normaldiet)withFigure10(highfatdiet)showsvariationwithboth biomarkerandtreatment.Forexamplewithnormaldiet(Figure9),triglycerides,HDL, advancedglycationendproductformaseparateclusterbutthesearenotclusteredwiththe highfatdiet(Figure10).Inthenormaldiet(Table9),butnotinthehighfatdiet(Figure10), DiaMetrixtendstoclusternearuntreated. Discussion ThestudyillustratedthebiochemicalactivityofDiaMetrixrelativetothecommonly usedantidiabeticdrugsagainstabackgroundofgeneticobesity.Analysisofthepatternsof cytokineandotherbiomarkersregulationprovidedinsightsintohowDiaMetrixexertsits salutaryeffectsindiabetesbysignificantlyregulatingexpressionofabroadrangeofsurrogate markersofdiabeticseverity. UsinganimalperformancestatusastheendpointweconcludedthatDiaMetrixand ByettawerebettertoleratedthanmetforminandActos.Increasedmortalityinthehighfat dietofDiaMetrixtreatedanimalsdidnotcomeasasurprise.Givenhighconcentrationof hydroxycitrateinDiaMetrixpreparation,feedingtheanimalswithlipidsasaprimaryfood sourceresultedinshuttingdownthetricarboxylicacidcycle.Infact,thisdemonstratedthe efficacyofDiaMetrixintheexperimentaldiabetesmodel. WenotedseveralsurprisingfindingsregardingDiaMetrixandotherdrugeffectson selectmetabolites,inparticularglucose,triglycerides,HDLandLDLplusVLDLcholesterol fractions,andADPandATPlevels,whichwerenotsignificantlyaffectedbyanytreatmenton normalorhighfatdiet.Theseresultsarelikelyanoutcomeoftheinvivomodelsystem,which sharesonlysomecharacteristicswithhumantype2diabetes(14). AnotherunexpectedfindinginthisstudywaslackofsignificanteffectofDiaMetrixon bloodglucoselevelsincontrasttothehumanstudy,inwhichweobservedarapiddropof bloodglucoseindiabeticpatientswithin30minutesofDiaMetrixadministration(Hampshireet al,manuscriptinpreparation).ThepotenthypoglycemicactivityofSyntre5isconsistentwith antiinflammatoryactivityoftheagent.Asproinflammatorycytokinesincreaseglucoselevels (15),otherproteinssuchasTNF orMIF (16)(17)cancauserapiddropinbloodglucoselevel withinminutesofadministration.Weneedtokeepinmindthatouranimalmodelofhuman type2diabetescannotbereadilyinterpolatedtohumandisease.Forexample,basedon knowndataonbloodglucoseinnormalmice(18),wefoundthattheanimalsattheendofthe studywerenormoglycemic,nothyperglycemic.Hypoglycemiaornormoglycemiaarenotin commonindiabetestype2(19),(20)anddiethasalargeeffectonbloodglucoselevels(21). Givenmajordifferencesindrugtreatment(shorttermintheclinicversuslongterminvivo)and limitationsoftheinvivomodel,theseapparentlycontradictoryfindingscanbeeasily reconciled.

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Significantintergroupdifferencesinexpressionlevelsofseveralmarkerswere dependentonthediet.Advancedglycationendproduct(AGE),whichisexemplifiedby glycatedhemoglobin(hemoglobinA1c),isformedinanonenzymaticpathwaybyexposureof plasmaproteinstohighlevelsofglucose(22).Proteinglycationisassociatedwith cardiovasculardisease,nephropathyandretinopathy.AlthoughDiaMetrixonnormaldietwas associatedwithincreasedadvancedglycationendproductconcentrations,asimilareffectwas alsoobservedforByetta.Sincethelevelsofadvancedglycationendproductareproportional notonlytoconcentrationofplasmaglucosebutalsotothehalflifeoftargetproteinsandare geneticallydetermined(23),itisconceivablethatbothDiaMetrixandByettamighthave affectedturnoverofglycatedproteinsinthisinvivomodel. Wefeelthatmetrics,suchasadvancedglycationendproductlevel,shouldbeconsidered moreintheframeofbeingdiseasesymptomsratherthanbeingelevatedtothestatusof diseaseand/ormechanismsofdisease.Thesemarkersshouldnotbeseenasleversthatcanbe graspedtoalterphysiologytheyaremorelikepressuregaugesonasteamengine.Blood sugarforinstancehasbeentwistedintothisstatus.Manythinkthatby'controlling'i.e. loweringbloodsugar,thatthediseaseiscontrolled.Yetevenwithperfectglucosecontrol therearestillproblems.Drugsthataredesignedtocontrolbloodsugarforinstancehavemany liabilitiessuchashypoglycemia.Thustheemphasisoncontrollingbloodsugarasasole endpointmaybemisplaced.Itismuchmoreimportanttolookatthecausesofdiabetes,i.e. theimmunesystem.Highbloodsugarshouldbeseenforwhatitisasymptomofdisease,not adiseaseinandofitself.Wearenotsaying,however,thathighbloodsugarisnotaproblem, becauseitis,butitisnotthewholeproblem.Therefore,thecytokineresultsareextremely importantinsupportingcontentionsabouttheeffectofthedrugonthedisease,notjustthe symptoms.Giventhattype2diabetesisfundamentallyaninflammatorydisease,theinsights gainedmayalsoguidedecisionsaboutcompositionofthedrug. Insulin,whichregulatesthelevelofbloodglucose,isstillproducedinthepancreasby theLangerhansislesintype2diabetes,howevertheresponseofcellsthroughoutthebodyis abnormal.Byreducingtheconcentrationofglucoseintheblood,insulinisthoughttoreduce thelongtermcomplicationsofdiabetes,includingdamagetothebloodvessels,eyes,kidneys andnerves(24).Inthisstudy,DiaMetrixinducedsignificanthypoinsulinemiaonnormaldiet. ThiscanbeviewedasabeneficialeffectofDiaMetrixconsideringthattheob/obmicewitha deficiencyintheleptinpathwayhaveelevatedinsulinlevels(25),(26). Pyruvatekinase,whichregulatestheratelimitingfinalstepofglycolysis,catalyzes conversionofphosphoenolpyruvatetopyruvate,whichentersthecitricacidcycleafter conversiontoacetylCoAandreactionwithoxaloacetatetoproducecitricacid.Indiabetesthe rateofglycolysisisdecreasedandsomeofthateffectisattributabletopyruvatekinase deficiency.Theassayableactivityofskeletalpyruvatekinasedoesnotalwaysreflectenzyme concentration(27).PyruvatekinaseisreversiblyinhibitedbyphosphorylationorATPalone.In

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additionlowbloodglucoseinducesphosphorylation.DiaMetrixelevatedpyruvatekinase activityonnormaldietwhileotherdrugshadnosignificanteffects.Increasedactivityof pyruvatekinaseupontreatmentwithDiaMetrixisunlikelytobeduetoreducedglucoseorATP levels,asthesemarkerswerenotsignificantlydifferentfromthecontrol.Ontheotherhand, longtermexposuretohydroxycitrate,oneofthemainingredientsofDiaMetrix,mayleadto upregulationofpyruvatekinaseexpression.Examplesofsuchdruginducedupregulation havebeenreported(28). Citrateisanintermediateinthetricarboxylicacidcycle,themainenergyproducing mechanisminacell.AfterthepyruvatedehydrogenasecomplexformsacetylCoAfrom pyruvateandcofactors(thiaminepyrophosphate,lipoamide,FAD,NAD+andCoA),citrate synthasecatalyzesthecondensationofoxaloacetatewithacetylCoAtoformcitrate.Citrate wassignificantlyincreasedbyDiaMetrixonlyonhighfatdiet,consistentwithinefficient tricarboxylicacidcycleoperationanddecreasedutilizationofcitrate. Inthepastfewyearsdiabeteshascometobeincreasinglyrecognizedasaninflammatory diseaseinwhichobesityplaysakeyroleinthemetabolicsyndromerelatedtoinsulinresistance andheightenedriskforthedevelopmentoftype2diabetes.Obesityisassociatedwithlow gradechronicinflammationassociatedwiththeadiposetissue.Inturn,theactivated inflammatorycascadecrosstalkswithinsulinsignalingpathwaysthroughinductionof cytokines,chemokinesandgrowthfactors(29). Circulatingbiomarkersoftheinflammatory pathway,ioncascade,endothelialdysfunction,andprocoagulantimbalancemaybeassociated withdevelopmentofbothtype1andtype2diabetes(21).Thecomorbiditiesofdiabetes,such asobesity,insulinresistance,hyperglycemia,hypertensionanddyslipidemia,furtheraggravate thenephropathy,retinopathyandcardiovasculardiseasewhileantihyperglycemictherapies relievethesesymptoms(30), (31). Remarkably,DiaMetrixsignificantlydownregulatedarangeofproinflammatory chemokines,cytokines,andgrowthfactors.Therolesofeotaxin(32),MCP1(3334),MCP3 (35)andMCSF(3637)inmaintenanceoftheinflammatorystatearewelldocumented.IL17 isofparticularimportance,astheIL23/IL17pathwayisakeyplayerinchronicinflammation andanumberofrelateddiseasesrecognizedascomorbiditiesofdiabetes(38),(39).GMCSFis amajorregulatoryfactorthatcontrolsthefunctionsofgranulocyteandmacrophagelineage populationsatallstagesofmaturationandplaysakeyroleininflammatoryandautoimmune diseases(40).TGFactinginconcertwithinflammatorycytokinessupportsdenovo differentiationofIL17producingTcells(41).Inaddition,DiaMetrixsuppressedcirculating levelsofanumberofimportantproinflammatorymarkersincludingthechemokineKCthatis homologoustohumanIL8(42),IL6(43),IFN (44)andVEGF(45)butalsoelevatedlevelsof antiinflammatorycytokineIL4(4647),yetanothertherapeuticallysignificanttarget.Mice expressingIL4inthepancreasareprotectedfromdiabetes(48),andtheadministrationof recombinantIL4preventsdiabetesindiabeticmice(49). Macrophageactivityisintimatelyconnectedtocytokinesandpancreaticisletscell deathintype2diabetes.Theisletsfrompatientswithtype2diabetesareinfiltratedby

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immunecellsthatproduceinflammatoryfactors(IL6,KC/IL8,GCSF,MIP1andMCP1). Increasedisletassociatedmacrophagesareobservedinhumantype2diabeticpatientsandin mostanimalmodelsofdiabetes,andinisletsofmiceonhighfatdietevenbeforetheonsetof diabetes(37).DiaMetrix,byvirtueofdownregulatingrelevantinflammatorymoleculesand upregulatingantiinflammatorymolecules,maybreakthisviciouscycleandslowdownthe processofisletcelldestruction. Sinceoneofthecharacteristicsofdiabetesisderangementoftheimmunesystem,and becausethemarkersmeasuredareallimmunesystemmessagesandeffectors,druginduced differencesfromcontrolareindicativeofdrugeffectsintreatmentgroupsandaredirectly implicatedinthemultiorgandysfunctionscharacteristicofdiabetes.Innormalimmune function,theinflammatorymarkerspeakfirstinresponsetosomestimulus,forinstanceinthe caseofinfection,subsequentlytheantiinflammatorymarkersstepinatalatertimetolimitthe damagecausedbytheproinflammatorymarkers.Inthiswaytheimmunesystemmobilizesto firstattackaninvaderandthencallsaceasefiretokeepcollateraldamagedown.Inmany autoimmunediseases,suchasdiabetes,thelinkageandcoordinationoftheproandanti inflammatorymechanismsseemstobebroken.Forinstancebothcanbeelevated simultaneously.Thiskindofdiscoordinationiscommoninmanyimmunemediateddiseases. Mechanistically,thefunctionsofcytokines,chemokinesandgrowthfactorsare reasonablywellunderstood.Manyproinflammatoryfactorsareviewedasnoveltherapeutic targetsforthetreatmentofchronicinflammatorydiseases,forexampleIL6(50),TNF (51), IL17,GMCSF(3840).DiaMetrixhasbeenshowntodownregulatethetwolattermarkers. Theimmunesystemanditseffectorsaretheprimarymediatorsofcellularandorgandamagein diabetes.Thusevaluatingtheeffectofthedrugontheimmunesystemwillallowustosaywith agreatdealofconfidencehowthedrugcausesitsbeneficialeffect.Theseresults,when consideredinlightoftheresultsofhistologicalexamination,supportthecontentionthat DiaMetrixmaybeaneffectiveinterventionfortype2diabetes. References
1. 2. 3. 4. 5. Kohn,L.D.,Wallace,B.,Schwartz,F.,andMcCall,K.(2005)Istype2diabetesanautoimmune inflammatorydisorderoftheinnateimmunesystem?,Endocrinology146,41894191. DeSouza,C.T.,Araujo,E.P.,Bordin,S.,Ashimine,R.,Zollner,R.L.,Boschero,A.C.,Saad,M.J., andVelloso,L.A.(2005)Consumptionofafatrichdietactivatesaproinflammatoryresponse andinducesinsulinresistanceinthehypothalamus,Endocrinology146,41924199. Howard,B.V.(2002)Dietaryfatasariskfactorfortype2diabetes,AnnNYAcadSci967,324 328. Han,D.H.,Hansen,P.A.,Host,H.H.,andHolloszy,J.O.(1997)Insulinresistanceofmuscle glucosetransportinratsfedahighfatdiet:areevaluation,Diabetes46,17611767. Oakes,N.D.,Cooney,G.J.,Camilleri,S.,Chisholm,D.J.,andKraegen,E.W.(1997)Mechanisms ofliverandmuscleinsulinresistanceinducedbychronichighfatfeeding,Diabetes46,1768 1774.

Page12of14 6. 7. 8. 9. 10. 11. 12. Venables,M.C.,andJeukendrup,A.E.(2009)Physicalinactivityandobesity:linkswithinsulin resistanceandtype2diabetesmellitus,DiabetesMetabResRev25Suppl1,S1823. Saenz,A.,FernandezEsteban,I.,Mataix,A.,Ausejo,M.,Roque,M.,andMoher,D.(2005) Metforminmonotherapyfortype2diabetesmellitus,CochraneDatabaseSystRev,CD002966. Lawrence,J.M.,andReckless,J.P.(2001)Actos(pioglitazone):anewtreatmentfortype2 diabetes,HospMed62,411416. Bond,A.(2006)Exenatide(Byetta)asanoveltreatmentoptionfortype2diabetesmellitus,Proc (BaylUnivMedCent)19,281284. Ozcan,U.,Cao,Q.,Yilmaz,E.,Lee,A.H.,Iwakoshi,N.N.,Ozdelen,E.,Tuncman,G.,Gorgun,C., Glimcher,L.H.,andHotamisligil,G.S.(2004)Endoplasmicreticulumstresslinksobesity,insulin action,andtype2diabetes,Science306,457461. Sharma,K.,McCue,P.,andDunn,S.R.(2003)Diabetickidneydiseaseinthedb/dbmouse,AmJ PhysiolRenalPhysiol284,F11381144. Goncalves,A.C.,Tank,J.,Diedrich,A.,Hilzendeger,A.,Plehm,R.,Bader,M.,Luft,F.C.,Jordan,J., andGross,V.(2009)Diabetichypertensiveleptinreceptordeficientdb/dbmicedevelop cardioregulatoryautonomicdysfunction,Hypertension53,387392. Mastropaolo,M.D.,Evans,N.P.,Byrnes,M.K.,Stevens,A.M.,Robertson,J.L.,andMelville,S. B.(2005)Synergyinpolymicrobialinfectionsinamousemodeloftype2diabetes,InfectImmun 73,60556063. http://www.taconic.com/wmspage.cfm?parm1=16. Kiely,A.,McClenaghan,N.H.,Flatt,P.R.,andNewsholme,P.(2007)Proinflammatorycytokines increaseglucose,alanineandtriacylglycerolutilizationbutinhibitinsulinsecretioninaclonal pancreaticbetacellline,JEndocrinol195,113123. Atsumi,T.,Cho,Y.R.,Leng,L.,McDonald,C.,Yu,T.,Danton,C.,Hong,E.G.,Mitchell,R.A.,Metz, C.,Niwa,H.,Takeuchi,J.,Onodera,S.,Umino,T.,Yoshioka,N.,Koike,T.,Kim,J.K.,andBucala,R. (2007)Theproinflammatorycytokinemacrophagemigrationinhibitoryfactorregulatesglucose metabolismduringsystemicinflammation,JImmunol179,53995406. SanchezZamora,Y.,Terrazas,L.I.,VilchesFlores,A.,Leal,E.,Juarez,I.,Whitacre,C.,Kithcart,A., Pruitt,J.,Sielecki,T.,Satoskar,A.R.,andRodriguezSosa,M.(2010)Macrophagemigration inhibitoryfactorisatherapeutictargetintreatmentofnoninsulindependentdiabetesmellitus, FASEBJ24,25832590. Wong,F.S.,andJaneway,C.A.,Jr.(1999)Insulindependentdiabetesmellitusanditsanimal models,CurrOpinImmunol11,643647. Murata,G.H.,Duckworth,W.C.,Hoffman,R.M.,Wendel,C.S.,Mohler,M.J.,andShah,J.H. (2004)Hypoglycemiaintype2diabetes:acriticalreview,BiomedPharmacother58,551559. DeFronzo,R.A.(2004)Pathogenesisoftype2diabetesmellitus,MedClinNorthAm88,787835, ix. http://www.taconic.com. Monnier,V.M.,andCerami,A.(1981)Nonenzymaticbrowninginvivo:possibleprocessfor agingoflonglivedproteins,Science211,491493. Leslie,R.D.,Beyan,H.,Sawtell,P.,Boehm,B.O.,Spector,T.D.,andSnieder,H.(2003)Levelof anadvancedglycatedendproductisgeneticallydetermined:astudyofnormaltwins,Diabetes 52,24412444. Lazarus,R.,Sparrow,D.,andWeiss,S.(1998)Temporalrelationsbetweenobesityandinsulin: longitudinaldatafromtheNormativeAgingStudy,AmJEpidemiol147,173179. Seufert,J.,Kieffer,T.J.,andHabener,J.F.(1999)Leptininhibitsinsulingenetranscriptionand reverseshyperinsulinemiainleptindeficientob/obmice,ProcNatlAcadSciUSA96,674679.

13.

14. 15.

16.

17.

18. 19. 20. 21. 22. 23.

24. 25.

Page13of14 26. Ishii,Y.,Ohta,T.,Sasase,T.,Morinaga,H.,Hata,T.,Miyajima,K.,Katusda,Y.,Masuyama,T., Shinohara,M.,Kakutani,M.,andMatsushita,M.(2010)Ahighfatdietinhibitstheprogression ofdiabetesmellitusintype2diabeticrats,NutrRes30,483491. Johnson,M.L.,andVeneziale,C.M.(1980)Concentrationofskeletalandcardiacmuscle pyruvatekinasedeterminedbyspecificradioimmunoassay,BiochimBiophysActa611,127135. Fatemi,S.H.,Reutiman,T.J.,Folsom,T.D.,Bell,C.,Nos,L.,Fried,P.,Pearce,D.A.,Singh,S., Siderovski,D.P.,Willard,F.S.,andFukuda,M.(2006)Chronicolanzapinetreatmentcauses differentialexpressionofgenesinfrontalcortexofratsasrevealedbyDNAmicroarray technique,Neuropsychopharmacology31,18881899. Oliver,E.,McGillicuddy,F.,Phillips,C.,Toomey,S.,andRoche,H.M.(2010)Theroleof inflammationandmacrophageaccumulationinthedevelopmentofobesityinducedtype2 diabetesmellitusandthepossibletherapeuticeffectsoflongchainn3PUFA,ProcNutrSoc69, 232243. Goldberg,R.B.(2009)Cytokineandcytokinelikeinflammationmarkers,endothelial dysfunction,andimbalancedcoagulationindevelopmentofdiabetesanditscomplications,J ClinEndocrinolMetab94,31713182. Kolb,H.,andMandrupPoulsen,T.(2010)Theglobaldiabetesepidemicasaconsequenceof lifestyleinducedlowgradeinflammation,Diabetologia53,1020. Crump,M.P.,Rajarathnam,K.,Kim,K.S.,ClarkLewis,I.,andSykes,B.D.(1998)Solution structureofeotaxin,achemokinethatselectivelyrecruitseosinophilsinallergicinflammation,J BiolChem273,2247122479. Lloyd,C.M.,Dorf,M.E.,Proudfoot,A.,Salant,D.J.,andGutierrezRamos,J.C.(1997)Roleof MCP1andRANTESininflammationandprogressiontofibrosisduringmurinecrescentic nephritis,JLeukocBiol62,676680. Conti,P.,andDiGioacchino,M.(2001)MCP1andRANTESaremediatorsofacuteandchronic inflammation,AllergyAsthmaProc22,133137. Tsou,C.L.,Peters,W.,Si,Y.,Slaymaker,S.,Aslanian,A.M.,Weisberg,S.P.,Mack,M.,andCharo, I.F.(2007)CriticalrolesforCCR2andMCP3inmonocytemobilizationfrombonemarrowand recruitmenttoinflammatorysites,JClinInvest117,902909. Oren,H.,Erbay,A.R.,Balci,M.,andCehreli,S.(2007)Roleofnovelbiomarkersofinflammation inpatientswithstablecoronaryheartdisease,Angiology58,148155. Ehses,J.A.,BoniSchnetzler,M.,Faulenbach,M.,andDonath,M.Y.(2008)Macrophages, cytokinesandbetacelldeathinType2diabetes,BiochemSocTrans36,340342. Hunter,C.A.(2007)Act1ivatingIL17inflammation,NatImmunol8,232234. Iwakura,Y.,andIshigame,H.(2006)TheIL23/IL17axisininflammation,JClinInvest116,1218 1222. Hamilton,J.A.(2002)GMCSFininflammationandautoimmunity,TrendsImmunol23,403408. Veldhoen,M.,Hocking,R.J.,Atkins,C.J.,Locksley,R.M.,andStockinger,B.(2006)TGFbetain thecontextofaninflammatorycytokinemilieusupportsdenovodifferentiationofIL17 producingTcells,Immunity24,179189. Neels,J.G.,Badeanlou,L.,Hester,K.D.,andSamad,F.(2009)Keratinocytederivedchemokine inobesity:expression,regulation,androleinadiposemacrophageinfiltrationandglucose homeostasis,JBiolChem284,2069220698. Wu,W.,Wang,M.,Sun,Z.,Wang,X.,Miao,J.,andZheng,Z.(2010)ThepredictivevalueofTNF alphaandIL6andtheincidenceofmacrovascularcomplicationsinpatientswithtype2 diabetes,ActaDiabetol.

27. 28.

29.

30.

31. 32.

33.

34. 35.

36. 37. 38. 39. 40. 41.

42.

43.

Page14of14 44. Hayashi,T.,Morimoto,M.,Iwata,H.,andOnodera,T.(1998)Interferongammaplaysarolein pancreaticisletcelldestructionofreovirustype2induceddiabeteslikesyndromeinDBA/1 sucklingmice,IntJExpPathol79,313320. Wang,J.,Xu,X.,Elliott,M.H.,Zhu,M.,andLe,Y.Z.(2010)MullercellderivedVEGFisessential fordiabetesinducedretinalinflammationandvascularleakage,Diabetes59,22972305. Zaccone,P.,Phillips,J.,Conget,I.,Gomis,R.,Haskins,K.,Minty,A.,Bendtzen,K.,Cooke,A.,and Nicoletti,F.(1999)Interleukin13preventsautoimmunediabetesinNODmice,Diabetes48, 15221528. Marie,C.,Pitton,C.,Fitting,C.,andCavaillon,J.M.(1996)Regulationbyantiinflammatory cytokines(IL4,IL10,IL13,TGFbeta)ofinterleukin8productionbyLPSand/orTNFalpha activatedhumanpolymorphonuclearcells,MediatorsInflamm5,334340. Gallichan,W.S.,Balasa,B.,Davies,J.D.,andSarvetnick,N.(1999)PancreaticIL4expression resultsinisletreactiveTh2cellsthatinhibitdiabetogeniclymphocytesinthenonobesediabetic mouse,JImmunol163,16961703. Tominaga,Y.,Nagata,M.,Yasuda,H.,Okamoto,N.,Arisawa,K.,Moriyama,H.,Miki,M.,Yokono, K.,andKasuga,M.(1998)AdministrationofIL4preventsautoimmunediabetesbutenhances pancreaticinsulitisinNODmice,ClinImmunolImmunopathol86,209218. Ding,C.,Cicuttini,F.,Li,J.,andJones,G.(2009)TargetingIL6inthetreatmentofinflammatory andautoimmunediseases,ExpertOpinInvestigDrugs18,14571466. Murdaca,G.,Colombo,B.M.,andPuppo,F.(2009)AntiTNFalphainhibitors:anewtherapeutic approachforinflammatoryimmunemediateddiseases:anupdateuponefficacyandadverse events,IntJImmunopatholPharmacol22,557565.

45. 46.

47. 48.

49.

50. 51.

Abbreviations GMCSF granulocytemacrophagecolonystimulatingfactor HFD highfatdiet ND normaldiet IL interleukin MCP1 monocytechemoattractantprotein1